The present invention relates to a blood-fluid composition, particularly, to a blood-fluid composition for use in a complement-mediated cell lysis system.
The following references are referred to by corresponding number herein:
1. Muller-Eberhard, H. J., Complement, Ann. Rev. Biochem., 44:697 (1975). PA0 2. Kataoka, T., Williamson, J. R., and Kinsky, S. C., Biochem. et Biophys. Acta, 298:158 (1973). PA0 3. Six, H. R., Uemura, K., and Kinsky, S. C., Biochemistry, 12(20):4003 (1973). PA0 4. Okada, N., Yasuda, T., Tsumita, T., and Okada, H., Immunology, 45:115 (1982). PA0 5. Yasuda, T., Naito, Y., Tsumita, T., and Tadakuma, T., J. Immonol. Methods, 44:153 (1981). PA0 6. Geiger, B., and Smolarsky, M., J. Immunol. Methods, 17:7 (1977). PA0 7. Uemura, K., Hattori, H., Kitazawa, N., and Taketomi, T., J. Immunol. Methods, 53:221 (1982). PA0 8. Stroud, R. M., Volanakis, J. E., and Lint, T. F., In: Immunochemistry of Proteins, edited by Atazzi, M. Z., Vol. 3, Plenum Press, N.Y., N.Y. (1979). PA0 9. Whicher, J. T., Clin. Chem., 24(1):7 (1978). PA0 10. Szoka, F., Jr., and Papahadjopoulos, D., Ann. Rev. Biophys. Bioeng., 9:467 (1980). PA0 11. Szoka, F., Jr. and Papahadjopoulos, D., Proc. Nat. Acad. Sci. U.S.A., 75:4194-4198 (1978). PA0 12. Heath, T. D., Macher, B. A. and Papanadjopoulos, D., Biochimica et Biophysica Acta, 640:66-81 (1981). PA0 13. Martin, F. J., Hubbell, W. L., and Papahadjopoulos, D., Biochemistry, 20:4229-4238 (1981). PA0 14. Martin, F. J. and Papahadjopoulos, D., J. Biol. Chem., 257:286-288 (1982). PA0 15. Bodmer, W. F., Tissue Antigens, 17:9 (1981).
The ability of complement to lyse lipid bilayer cells, such as red blood cells (reference 1) and liposomes (reference 2-4), in the presence of ligand-specific cell surface binding agents has been exploited in immunoassay methods for determination of ligand or anti-ligand analytes. U.S. Pat. No. 4,130,634 to Molinaro, et al. describes a method for detecting erythrocyte-specific antigens by coating the erythrocytes with anti-antigen antibody in the presence of complement, and measuring the release of hemoglobin from the lysed cells. Yasuda, et al. have described a simple method for measuring anti-glycolipid antibody by reacting the antibody with complement and liposomes containing surface glycolipid, where cell lysis and release of a fluorogenic reporter from the liposomes produces increased fluorescence in the assay medium (reference 5). Similar types of liposome immunoassays applicable to ganglioside GM.sub.2 antigen (reference 6), and to Forsmann and blood group A-active gangliolipids (reference 7) have been reported.
Several types of reporters, in addition to chromophores, such as hemoglobin, and fluorogenic compounds, have been used to indicate cell lysis in immunoassays of this type. U.S. Pat. No. 3,887,698 to McConnell, et al. discloses a liposome immunoassay test system in which the difference in electron paramagnetic resonance spectra between encapsulated and released nitroxide reporter provides a measure of complement-mediated liposome lysis. U.S. Pat. Nos. 4,235,792 to Hsia, et al. and 4,342,826 to Cole disclose liposome immunoassay systems in which complement-mediated cell lysis is evidenced by the expression of liposome-encapsulated enzymes.
Immunoassay systems involving lysable lipid membrane vesicles provide important potential advantages in diagnostic test systems. One advantage is that the ligand/anti-ligand binding reaction and the measurement of released reporter from lysed cells can be performed in the same assay mixture. This single-mixture assay, which is generally referred to as a homogeneous assay, contrasts with more widely used enzyme or fluorescent immunoassays which require the steps of (1) binding a ligand-reporter complex to a solid surface in the presence of analyte in one mixture, (2) removing unbound ligand-reporter complex, and (3) measuring bound or unbound reporter levels in a second mixture. Homogeneous cell-lysis immunoassays also have the potential for high assay sensitivity, since relatively few ligand/anti-ligand binding events on the cell surface can lead to the release or expression of a large number of reporter molecules.
Heretofore, however, the reliability and sensitivity of homogeneous immunoassays have been seriously limited by variable, and in many cases prohibitively high, non-specific lysis which occurs when lysable target cells are mixed with a serum source of complement and/or a serum source of analyte. Studies conducted in support of the present application, reported below, show that serum added to a cell lysis system causes non-specific cell lysis to levels as high as 50%-70% of total releasable marker. The degree of non-specific lysis can depend on several variables, including the age, source and storage conditions of the serum, the age, lipid composition and storage conditions of the lysable target cells, and the relative concentrations of serum and lysable cells.
The causative serum factors which produce non-specific cell lysis likely include cross reactive serum antibodies, such as immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies capable of cross-reacting with cell-surface ligands to activate the classical complement pathway. It is also known that certain lipids, polynucleotides, C-reactive proteins and some viruses can stimulate the classical complement pathway. Another source of interfering factor in serum may include serum components such as polysaccharides, lipopolysaccharides and immunoglobulins which are capable of activating the alternative complement pathway. (References 1, 8 and 9 provide excellent reviews of the complement system). That one or both complement pathways are responsible for some observed non-specific lysis is supported by experiments conducted in conjunction with the present application showing that inactivation of complement components, either upon short-term storage or by heat-treating the serum, results in an appreciable decrease in the level of non-specific cell lysis. The just mentioned experiments also indicate that heat-treated serum retains substantial non-specific cell lytic activity, indicating that the serum contains yet a third type of interference factor which is distinguished by its ability to induce cell lysis via non-complement mechanism(s). It is noted that even if heat-treating serum could effectively eliminate non-specific cell-lysis, the requirement for active complement in a homogeneous assay system would preclude this approach.
It is one general object of the invention to provide a blood composition which substantially overcomes problems in variability and high non-specific lysis associated with complement-mediated cell lysis systems known in the prior art.
Another object of the invention is to provide such a composition for use in a homogeneous assay which is based on ligand-specific, complement-mediated cell lysis.
A related object of the invention is to provide, for use in such an assay system, a blood-fluid composition which substantially enhances the reliability of the assay, and increases the signal-to-noise ratio thereof generally between about 4- and 10-fold.
It is yet another object of the invention to provide such a composition which is easily and inexpensively prepared as part of a homogeneous cell lysis immunoassay.
A further object of the invention is to provide an improved complement preparation for use in a ligand-specific, complement-mediated cell lysis system.
Other objects of the invention include providing a homogeneous assay system and specific homogeneous assay kits utilizing such blood fluid composition, and providing a method for treating blood fluid to reduce non-specific lysis in a complement-mediated cell-lysis system.
The present invention includes a blood fluid composition adapted to be added to a complement-mediated cell-lysis system. The composition includes the blood fluid and lipid vesicles which, when added to the system, are capable of increasing the ratio of ligand-specific cell lysis occurring in the presence of an added anti-ligand, to non-specific lysis, occurring in the absence of added anti-ligand. The vesicles are added to the blood fluid in an amount which produces at least a 2-fold increase in the specific to non-specific ratio over that achievable in the system under the same conditions in the absence of the vesicles. In a preferred embodiment of the invention, the vesicles include liposomes predominantly in the 0.02 to 0.2 micron size range, and the vesicles are added in an amount which enhances such ratio between about 4- and 10-fold.
According to one aspect of the invention, the blood fluid composition forms part of a homogeneous assay system for the determination of ligand-related analyte, and in particular, an analyte present in a serum or plasma sample. Here the blood fluid may include both a serum source of complement and the analyte-containing serum sample. The lipid vesicles added to the system are effective in reducing non-specific lysis due to both serum sources.
A kit for the determination of a ligand-related analyte includes lysable cells, a serum source of complement, and such lipid vesicles. Also dislcosed is a method for treating blood fluid, such as serum or plasma, to reduce significantly the extent of non-specific lysis produced when the blood fluid is added to a cell-lysis system containing lysable cells.
These and other objects and features of the present invention will become more fully apparent from the following detailed description of the invention.